5-aza-2-deoxycytidine (DAC) is approved for the treatment of myelodysplastic syndromes, but resistance to this agent is common. In search for mechanisms of resistance, we measured the half maximal (50%) inhibitory concentration (IC 50 ) of DAC and found it differed 1000-fold among a panel of cancer cell lines. The IC 50 was correlated with the doses of DAC that induced the most hypomethylation of long interspersed nuclear elements (LINE; R ؍ 0.94, P < .001), but not with LINE methylation or DNA methyltransferase 1 (DNMT1), 3a, and 3b expression at baseline. Sensitivity to DAC showed a low correlation (R ؍ 0.44, P ؍ .11) to that of 5-azacytidine (AZA), but a good correlation to that of cytarabine (Ara-C; R ؍ 0.89, P < .001). The 5 cell lines most resistant to DAC had a combination of low dCK, hENT1, and 2 transporters, and high cytosine deaminase. In an HL60 clone, resistance to DAC could be rapidly induced by drug exposure and was related to a switch from heterozygous to homozygous mutation of DCK. Transfection of wild-type DCK restored DAC sensitivity. DAC induced DNA breaks as evidenced by H2AX phosphorylation and increased homologous recombination rates by 7-to 10-fold. These results suggest that in vitro resistance to DAC can be explained by insufficient incorporation into DNA. (Blood. 2009;113:659-667)
DNA methylation is commonly thought of as a "molecular lock" that leads to permanent gene silencing. To investigate this notion, we tested 24 different HDAC inhibitors (HDACi) on colon cancer cells that harbor a GFP locus stably integrated and silenced by a hypermethylated CMV promoter. We found that HDACi efficiently reactivated expression of GFP and many other endogenous genes silenced by DNA hypermethylation. After treatment, all promoters were marked with active chromatin, yet DNA hypermethylation did not change. Thus, DNA methylation could not prevent gene reactivation by drug-induced resetting of the chromatin state. In evaluating the relative contribution of DNA methylation and histone modifications to stable gene silencing, we followed expression levels of GFP and other genes silenced by DNA hypermethylation over time after treatment with HDACi or DNA demethylating drugs. Reactivation of methylated loci by HDACi was detectable for only 2 weeks, whereas DNA demethylating drugs induced permanent epigenetic reprogramming. Therefore, DNA methylation cannot be considered as a lock for gene expression, but rather as a memory signal for long-term maintenance of gene silencing. These findings define chromatin as an important druggable target for cancer epigenetic therapy and suggest that removal of DNA methylation signals is required to achieve long-term gene reactivation.
PurposeThe DNA methylation inhibitor 5-aza-2′-deoxycytidine (DAC) is approved for the treatment of myelodysplastic syndromes (MDS), but resistance to DAC develops during treatment and mechanisms of resistance remain unknown. Therefore, we investigated mechanisms of primary and secondary resistance to DAC in MDS.Patients and MethodsWe performed Quantitative Real-Time PCR to examine expression of genes related to DAC metabolism prior to therapy in 32 responders and non-responders with MDS as well as 14 patients who achieved a complete remission and subsequently relapsed while on therapy (secondary resistance). We then performed quantitative methylation analyses by bisulfite pyrosequencing of 10 genes as well as Methylated CpG Island Amplification Microarray (MCAM) analysis of global methylation in secondary resistance.ResultsMost genes showed no differences by response, but the CDA/DCK ratio was 3 fold higher in non-responders than responders (P<.05), suggesting that this could be a mechanism of primary resistance. There were no significant differences at relapse in DAC metabolism genes, and no DCK mutations were detected. Global methylation measured by the LINE1 assay was lower at relapse than at diagnosis (P<.05). On average, the methylation of 10 genes was lower at relapse (16.1%) compared to diagnosis (18.1%) (P<.05).MCAM analysis showed decreased methylation of an average of 4.5% (range 0.6%–9.7%) of the genes at relapse. By contrast, new cytogenetic changes were found in 20% of patients.ConclusionPharmacological mechanisms are involved in primary resistance to DAC, whereas hypomethylation does not prevent a relapse for patients with DAC treatment.
The DNA hypomethylating drug decitabine (DAC) reactivates silenced gene expression in cancer and is approved for the treatment of the myelodysplastic syndrome. Gene reactivation after DAC is variable and incompletely understood. Here, we established a cell line system (YB5) derived from the SW48 colon cancer cell line to study DAC-induced reactivation. YB5 contains a hypermethylated cytomegalovirus promoter driving green fluorescent protein (GFP), and the locus is transcriptionally silent. GFP reexpression can be achieved by DAC treatment, but the expression level of individual cells is heterogeneous. DAC-treated YB5 cells were separated into GFP-positive and GFP-negative subpopulations. By comparing DAC-treated sorted GFP-positive and GFP-negative cells, we found that their methylation levels were similarly decreased but that histone modifications and histone H3 densities were remarkably different. Despite a similar degree of (incomplete) DNA hypomethylation, GFP-positive cells reverted to an active chromatin structure marked by higher H3K9 acetylation, lower H3K27 trimethylation, and lower promoter nucleosome density. GFP-negative cells had histone modifications and promoter nucleosome density, similar to parental cells. On DAC withdrawal, gradual resilencing and remethylation occurred in both GFP-positive and GFP-negative cells, and the resilencing correlated with a gradual increase in nucleosome occupancy in GFP-positive cells. These data show that hypomethylation alone after DAC is insufficient for gene expression induction, and that chromatin resetting to an active state including nucleosome eviction is required for activation of protein expression. Our findings suggest that gene expression is the key in optimizing DAC treatment strategies in the clinic.
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